To investigate the effects of mosquitofish, Gambusia affinis on aquatic insect biodiversity in South China, three common species of aquatic insects in mosquitofish habitat, i.e., Cloeon bengalense, Chironomus kiiensis and Aedes albopictus, were selected under the lab condition to test the feeding preference of mosquitofish. The resulted showed that mosquitofish had no significant feeding selection on all preys in low density under the single-predator-and-multiple-preys experimental design. But mosquitofish preferred the larvae and pupa of A. albopictus to other preys with the increase of prey density. When the prey ratio was 1∶1 under the single-predator-and-two-prey experimental design, mosquitofish frequently prefer larvae of A. albopictus to the other prey. However, there was no significant influence to total prey consumption for mosquitofish when prey ratio was changed but total prey numbers unchanged. The results suggested that G. affinis preferred to feed on the mobile prey close to water surface and showed no interest in the unmoving or dead prey, and mosquito larvae were the favorite prey of mosquitofish compared with mayflies and chironomids.